Polymyxin B Analogs for LPS Detoxification

ABSTRACT

The invention relates to SAEP II peptide dimers that mimic polymyxin B i.a. in its ability to bind non-covalently the lipopolysaccharide (LPS) of Gram-negative bacteria with high affinity, and therefore to detoxify LPS as polymyxin B does. The dimeric structure is maintained by a pair of disulphide bonds involving the two cystein residues present in the peptide sequence, which does not exceed 17 amino acids and essentially comprises cationic and hydrophobic amino acid residues. In the dimers of the invention, peptides may have a parallel or anti-parallel orientation. As a matter of example, a dimer of the invention is constituted by a peptide of formula NH2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH, either in a parallel or antiparallel dimeric form. SAEP II dimers are useful for treating or preventing septic shock and related disorders generated by Gram-negative bacteria infection. The invention also relates to LPS-peptide complexes in which LPS and SAEP II dimers are non-covalently bound together. These complexes are useful as vaccinal agents against Gram-negative bacteria infection.

The present invention relates to peptide analogs of polymyxin B that areuseful for LPS detoxification. In the pharmaceutical field, they may beused (i) as such i.a. to treat fatal disorders, such as septic shock,caused by Gram-negative bacteria infection; or (ii) non-covalently boundto LPS which is therefore detoxified; the complex thereof being usefulas vaccinal agent against Gram-negative bacteria infection.

Lipopolysaccharide (LPS) is a major constituent of the outer membrane ofthe cell wall of Gram-negative bacteria. LPS is highly toxic in mammals,particularly humans and with regard of its biological activity has beencalled endotoxin. It is responsible for the effects deriving fromendotoxicosis in septic shock, a life-threatening event that occurs uponacute infection (sepsis) by Gram-negative bacteria.

LPS structure is constituted by a lipid moiety, called Lipid A,covalently linked to a polysaccharide moiety.

Lipid A is responsible for the toxic effect of LPS, in particularthrough interaction with B-cells and macrophages. This interactioninduces the secretion of pro-inflammatory cytokines. The inflammatorycondition may reach the fatal state of endotoxic shock.

Lipid A is highly hydrophobic and anchors LPS in the outer layer of thebacterial cell wall. Lipid A is composed of (i) a conservedbis-phosphorylated disaccharide region (most frequently, N,O-acylbeta-1,6-D-glucosamine 1,4′-bisphosphate) with (ii) fatty acids, thatsubstitute various hydrogen atoms pertaining to the disaccharidehydroxyls. The number of the fatty acids and their composition areinterspecies variable. As a matter of example, each of the two symmetricglucosamines (GlcN1 and GlcN2) of Neisseria meningitidis lipid A carriesthe following fatty acids: 2N—C14,3OH; C12; and 3O—C12,3OH.

The LPS polysaccharide moiety is constituted by carbohydrate chains,responsible for antigenicity. The carbohydrate chain structure is itselfcomposed of (i) a conserved inner core called the KDO (2-keto, 3-desoxyoctulosonic acid) region bound to lipid A and (ii) a variable outer corebound to the KDO region, that is commonly defined as including varioussaccharides, and the first repeat unit (that may comprised up to tensaccharides) of (iii) the external O-specific chains

In Gram-negative, non-enteric bacteria such as Neisserias, Bordetellas,Haemophilus and Moraxellas, the O-specific chains do not exist (what isdefined as the first repeat unit is in fact not repeated). Therefore,the LPS of these bacteria are often referred to as lipooligosaccharide(LOS).

LPS is not only toxic but also highly immunogenic. In mammals, anti-LPSantibodies are induced during infection and carriage, and may beprotective. In view of this, it has been already proposed to detoxifyLPS and to use the detoxified form thereof in prophylaxis ofGram-negative bacterial infections and related diseases.

Several detoxification methods are already known. In particular, it ispossible to detoxify LPS while using polymyxin B or more appropriately,peptide analogs thereof.

Polymyxin B is a molecule that binds Lipid A with high affinity so thatLPS is significantly detoxified. When given therapeutically in animalmodels, polymyxin B can prevent septic shock. However, polymyxin B is apolycationic antibiotic that may be somewhat toxic to humans because ofits non-biodegradability and the consequent tendency to accumulate inthe kidneys. Therefore, it is not recommended for use in prophylactic ortherapeutic products.

To overcome this limitation, peptide analogs to polymyxin B have beendeveloped. They do not retain the polymyxin B toxicity but merely mimicthe primary and secondary structures of polymyxin B and bind lipid A atthe same site as polymyxin B does, so that a LPS-peptide complex isformed. As a result, LPS is detoxified. Peptide analogs are inparticular described in U.S. Pat. No. 5,358,933, WO 93/14115, WO95/03327, WO 96/38163, EP 842 666 and EP 976 402. One of them, thecyclic monomer SAEP2 (synthetic anti-endotoxin peptide 2) of formulaKTKCKFLKKC has been more particularly studied (Rustici et al, 1993,Science 259: 361 and Velucchi et al, 1997, J. Endotox. Res. 4(4): 261).

It has now been found that the SAEP2 peptide as well as similar peptidesincluding in their sequences a number of uncharged polar amino acidssurrounded by two adjacent cysteine residues and counter-balanced by ashort external tail made of cationic amino acids (hereinaftergenerically referred to as SAEP II peptides) are of particular interestwhen they are in dimeric form; the dimer being conformationally made andmaintained by a pair of disulphide bonds between the cysteine residues.Indeed, SAEP II peptide dimers exhibit enhanced detoxificationproperties over the corresponding monomers.

Therefore, the invention relates to a SAEP II peptide dimer of formula(I)

NH2-A-Cys1-B-Cys2-C-COOH

NH2-A′-Cys1-B′-Cys2-C′-COOH

-   -   wherein the two Cys1 residues are linked together through a        disulphide bond and the two Cys2 residues are linked together        through a disulphide bond;        or formula (II)

NH2-A-Cys1-B-Cys2-C-COOH

COOH-C′-Cys2-B′-Cys1-A′-NH2

-   -   wherein the Cys1 residues are linked to the Cys2 residues        through a disulphide bond;        wherein A and A′ independently are a peptide moiety of from 2 to        5, preferably 3 or 4 amino acid residues, in which at least 2        amino acid residues, are independently selected from Lys, Hyl        (hydroxy-Lysine), Arg and His;        wherein B and B′ independently are a peptide moiety of from 3 to        7, preferably 4 or 5 amino acid residues, which comprise at        least two, preferably three amino acid residues independently        selected from Val, Leu, Ile, Phe, Tyr and Trp; and        wherein C and C′ are optional (these positions may be empty or        not) and are independently an amino acid residue or a peptide        moiety of from 2 to 3 amino acid residues;

provided that the cationic amino acid residues/hydrophobic amino acidresidues ratio (cat/hydroph ratio) is from 0.4 to 2, advantageously from0.5 to 1.2 or 1.5, preferably from 0.6 to 1; most preferably from 0.6 to0.8; e.g. 0.75.

Advantageously, A and A′ independently are a peptide moiety of from 2 to5, preferably 3 or 4 amino acid residues, in which at least one,preferably 2 amino acid residues, are independently selected from Lys,Hyl, Arg and His; those that are not selected from Lys, Hyl, Arg and His(“the remaining amino acid residues”), if any, being selected from thegroup consisting of uncharged polar or nonpolar amino acids residues;preferably Thr, Ser and Gly; most preferably Thr.

When the A and A′ peptide moieties comprise 3 amino acid residues, eachof them can be a cationic residue; or alternatively, two out of threeresidues are cationic amino acids, whereas the remaining residue isselected from the group consisting of uncharged polar or nonpolar aminoacids residues; preferably Thr, Ser and Gly; most preferably Thr.

When the A and A′ peptide moieties comprise 4 amino acid residues, it ispreferred that two or three out of four residues be selected from thegroups of cationic amino acid residues as defined above, whereas theremaining residue(s) is (are) selected from the group consisting ofuncharged polar or non-polar amino acids residues as defined above.

When the A and A′ peptide moieties comprise 5 amino acid residues, it ispreferred that three or four out of five residues be selected from thegroups of cationic amino acid residues as defined above, whereas theremaining residue (s) is (are) selected from the group consisting ofuncharged polar or non-polar amino acids residues as defined above.

Advantageously, B and B′ independently are a peptide moiety of from 3 to7, preferably 4 or 5 amino acid residues, which comprises at least two,preferably three amino acid residues independently selected from Val,Leu, Ile, Phe, Tyr and Trp; preferably from Leu, Ile and Phe; those thatare not selected from Val, Leu, Ile, Phe, Tyr and Trp (“the remainingamino acid residues”), if any, being independently selected from thegroup consisting of Lys, Hyl, Arg and His. As may be easily understood,the B and B′ peptide moieties may comprise up to 7 amino acid residuesindependently selected from Val, Leu, Ile, Phe, Tyr and Trp.

Advantageously, the B and B′ peptide moieties comprise the sequence-X1-X2-X3-, in which X1 and X2; X2 and X3; or X1, X2 and X3 areindependently selected from Val, Leu, Ile, Phe, Tyr and Trp; preferablyfrom Leu, Ile and Phe. In a preferred embodiment, the sequence-X1-X2-X3- comprises the Phe-Leu motif.

Particular embodiments of peptide moieties B and B′ include:

(i) the -X1-2-X3- sequence in which:

-   -   X1 is Lys, Hyl, His or Arg, preferably Lys or Arg; more        preferably Lys;    -   X2 is Phe, Leu, Ile, Tyr, Trp or Val; preferably Phe or Leu;        more preferably Phe; and    -   X3 is Phe, Leu, Ile, Tyr, Trp or Val; preferably Phe or Leu;        more preferably Leu; and        (ii) amino acid residues, if any, each being independently        selected from the group consisting of Val, Leu, Ile, Phe, Tyr,        Trp, Lys, Hyl, Arg and His; preferably Val, Leu, Ile, Phe, Tyr        and Trp; more preferably Leu, Ile and Phe.

When B and B′ comprise more than 4 nonpolar amino acid residues, A andA′ preferably comprises at least 3 positively charged amino acidresidues.

In the C and C′ peptides moieties, the amino acid residue(s) may be anyamino acid residues provided that the cationic amino acidresidues/hydrophobic amino acid residues ratio remains within thespecified range. Advantageously, they are independently selected fromuncharged amino acid residues polar or nonpolar, these latter beingpreferred. However, in a preferred manner, C and C′ are empty positions.

Therefore, a preferred class of dimers are of formula (III)

NH2-A-Cys1-B-Cys2-COOH

NH2-A′-Cys1-B′-Cys2-COOH

or formula (IV)

NH2-A-Cys1-B-Cys2-COOH

HOOC-Cys2-B′-Cys1-A′-NH2

wherein A, A′, B and B′ are as described above; provided that thecationic amino acid residues/hydrophobic amino acid residues ratio isfrom 0.4 to 2, advantageously from 0.5 to 1.2 or 1.5, preferably from0.6 to 1; most preferably from 0.6 to 0.8; e.g. 0.75.

Dimers of formula (I) or (III), that is with peptides in the parallelorientation, are referred to as parallel dimers. Dimers of formula (II)or (IV), that is with peptides in the anti-parallel orientation, arereferred to as antiparallel dimers.

In formulas (I) to (IV), A and A′ are preferably identical. The sameholds true for B and B′; and C and C′. A peptide dimer of formula (I),(II), (III) or (IV), in which A and A′; B and B′; and C and C′ aretwo-by-two identical, is referred to as homologous dimer. Indeed, inthis case, the peptide subunits included in the dimer are identical.

As a matter of example, the following peptides are cited as beingsuitable for use in dimers of the invention:

NH₂-Lys-Arg-His-Hyl-Cys-Lys-Arg-Ile-Val-Leu-Cys-COOH;

NH₂-Lys-Arg-His-Cys-Val-Leu-Ile-Trp-Tyr-Phe-Cys-COOH;

NH₂-Lys-Thr-Lys-Cys-Lys-Phe-Leu-Leu-Leu-Cys-COOH; and

NH₂-Hyl-Arg-His-Lys-Cys-Phe-Tyr-Trp-Val-Ile-Leu-Cys-COOH.

The respective cat/hydroph ratio of the corresponding homologous dimersare 2.00, 0.50, 0.75 and 0.67.

A particular example of the dimers described above, is constituted by apeptide of formula (V)NH2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH. This peptide ishereinafter referred to as the SAEP2-L2 peptide. As described above, itcan also be in parallel or anti-parallel dimeric form.

Peptides involved in the or dimers of the invention can beconventionally synthesized by classical methods using e.g. acomputer-driven automatic synthesizer. It is within the skills ofprofessional practitioners in the art of peptide synthesis to know howto design procedures so that a particular peptide is obtained. It goeswithout saying that during the synthesis phase, the cysteine thiolgroups can be protected. Once the synthesis is completed, they arede-protected and oxidation of the thiol groups is achieved in order togenerate the cyclic monomer, the parallel or anti-parallel dimer.

When both cysteine residues present in the peptide are de-protectedsimultaneously, it is theoretically possible to generate each of thethree forms upon oxidation. Then each of the three forms can beseparated from each other by conventional biochemical purificationmethods. Preparative reverse-phase high performance liquidchromatography (RP-HPLC) is cited as a suitable example. Indeed, one mayexpect that each of the three forms elutes at a different retentiontime. Therefore, a preparation containing the purified cyclic monomer,or the purified parallel and anti-parallel dimers can be simply obtainedby pooling together the respective peak fractions.

The respective proportions of each of the three forms generated uponoxidation depend on i.a. the specific amino acid sequence andimportantly, the concentration of the peptide. It may happen that one ortwo of the three forms be predominantly created and indeed, theprevalence of one or two forms may be such that the other(s) are notformed at all.

As a matter of example, the SAEP2-L2 peptide spontaneously oxidises intocyclic monomer and anti-parallel dimer, in proportions, which dependfrom the concentration of the peptide in solution. The internal sterichindrance of the “side-chains” (the NH2-Lys-Thr-Lys-portion) of theanti-parallel dimer is obviously lower than that of the parallel dimerand one may expect that a lower minimal energy be responsible for theprivileged formation of the anti-parallel dimer in aqueous solvents bycomparison with the parallel dimer. As a direct consequence of thisconcentration-driven process, the formation of the anti-parallel dimerand to a lesser extent the cyclic monomer is favoured up to theexclusion of the parallel dimer from the equilibrium.

When the parallel dimer cannot be spontaneously generated uponoxidation, it is necessary to adopt particular measures to make thepeptide associate within the parallel orientation. These measures arewithin the skills of the professional practitioners in the art ofpeptide synthesis. Nevertheless and as a matter of example only, it isindicated that differential protection of the Cys1 and Cys2 amino acidsfollowed by selective de-protection is a convenient way to achievedimerisation with the parallel orientation. Then the dimer may bepurified by conventional methods, including RP-HPLC.

Peptides that are chemically synthesized and purified are commonlyobtained in salt form due to the fact that acids and salts are usedduring the chemical synthesis and purification steps. Acetate is a saltcommonly used. Therefore, it shall be understood that the term “peptide”as used in the present description encompasses the salt form as well.

Peptides for use in the dimers of the invention can be characterized byvarious techniques, including i.a. Ion Cyclotron Resonance (ICR), MassAssisted Laser Desorption Ionisation-Time of Flights (MALDI-ToF)spectrometry and Nuclear Magnetic Resonance (NMR) spectrophotometry. Inparticular, it is possible to discriminate each of the three forms(cyclic monomer, parallel and anti-parallel dimer) by NMR analysis.MALDI-ToF mass spectrometry allows discriminating between monomer anddimers only.

The purity of compounds of the invention can be evaluated by RP-HPLC.Briefly, a preparation of compound is submitted to RP-HPLC. The relativepurity degree is calculated by integrating the peak surfaces. It isexpressed as the compound peak surface/surfaces of the whole peaks. Itis usual to prepare compounds of the invention that each exhibits apurity degree of at least 95%, frequently of at least 97%.

The invention also relates to compositions comprising:

-   -   A SAEP II peptide, wherein the peptide is essentially in dimeric        parallel form;    -   A SAEP II peptide, wherein the peptide is essentially in dimeric        anti-parallel form; or    -   mixtures thereof.

By “essentially” it is meant that in the compositions, a particular formis at least 95%, preferably at least 97%, more preferably 98% pure.

Mixed compositions in which the SAEP II peptide is present under severalforms (dimeric parallel, dimeric anti-parallel and/or monomeric forms)may spontaneous result from the evolution of a composition comprising asingle entity, e.g. the dimeric parallel form, kept at an appropriatetemperature over a certain period of time. This may be revealed by e.g.RP-HPLC analysis. The respective amounts of the various peptide formsmay be quantified by the same token.

The SAEP II dimers are useful as such as a detoxifying agent ofGram-negative bacterial LPS in vitro as well as in vivo. Accordingly,they may be used to prevent or treat pathological conditions due to therelease of LPS into the systemic circulation, e.g. into blood, as aresult of Gram-negative bacteria infections. These conditions includei.a. endotoxicosis, bacterial sepsis and septic shock.

Therefore, the invention encompasses:

-   -   The pharmaceutical use of a compound or composition of the        invention;    -   A pharmaceutical composition comprising a compound or a        composition of the invention together with a pharmaceutically        acceptable diluent or carrier;    -   The use of a compound or composition of the invention in the        preparation of a medicament for treating or preventing septic        shock; and    -   A method for treating or preventing septic shock, which        comprises administering a therapeutically or prophylactically        effective amount of a compound or composition of the invention,        to an individual in need.

A compound or composition of the invention may be administered tomammals, i.e. humans, when a Gram-negative bacteria infection isdiagnosed that may lead to endotoxicosis, bacterial sepsis and/or septicshock. Gram-negative bacteria that may be responsible for these fataldisorders include i.a., N. meningitidis, E. coli, Salmonella typhi,Bordetella pertussis and Pseudomonas aeruginosa. A compound orcomposition of the invention may be administered to an individual inneed by a systemic route, preferably the intravenous route. The dose tobe administered depends on various factors including i.a. the age,weight, physiological condition of the patient as well as the infectionstatus. It may be administered once or several times until the risk offatal event is avoided.

Since the SAEP II dimers and the SAEP2-L2 peptide are also able todetoxify LPS in vitro, the invention also relates to a LPS-peptidecomplex comprising (i) a LPS moiety of Gram-negative bacteria, and (ii)a SAEP II peptide dimer or the SAEP2-L2 peptide; wherein the LPS moietyand the SAEP II peptide dimer or the SAEP2-L2 peptide are non-covalentlybound to each other.

LPS detoxification may be assessed in a number of assays referred to inthe European Pharmacopeia They include the Limulus Amebocyte Lysate(LAL) assay; the pyrogen test in rabbits and the acute toxicity assay inD-galactosamine sensitized mice. These assays are illustratedhereinafter in the examples. In each of the assays the effect of LPS andthat of the LPS-peptide complex are measured in parallel so that adetoxification ratio be established.

In the LAL assay, the detoxification ratio is expressed by theLPS/LPS-peptide complex ratio. In the pyrogen test and the acutetoxicity assay, the detoxification ratio is expressed by the LPS-peptidecomplex/LPS ratio.

Significant detoxification is achieved, when the detoxification ratiomeasured in:

-   -   (i) the LAL assay is at least of 100, preferably 500, more        preferably 1000;    -   (ii) the pyrogen test is at least of 50, preferably of 100, more        preferably 500; or    -   (iii) D-galactosamine mice is at least of 50, preferably of 100,        more preferably of 200.

Detoxification may also be evaluated while comparing the effect of LPSand a LPS-peptide complex on the release of pro-inflammatory cytokinessuch as IL6, IL8 and TNFα, in in vitro or in vivo assays. These assaysare illustrated hereinafter in the examples. Significant detoxificationis achieved, when the LPS-peptide complex allows for at least 25-folddecrease, preferably at least 50-fold, more preferably at least 75-fold,most preferably at least 100-fold decrease in IL6 secretion in the invivo assay as described in the examples, section 5.4.1.

LPS-peptide complex of the invention is advantageously characterized bya molar LPS:peptide ratio of from 1:1.5 to 1:0.5, preferably 1:1.2 to1:0.8, more preferably of 1:1.1 to 1:0.9, most preferably 1:1.

For use in the complex of the invention the LPS is advantageously a LPSof N. meningitidis; E. coli; Salmonella typhi; Salmonella paratyphi;Shigella flexneri Haemophilus influenzae; Helicobacter pylori; Chlamydiatrachomatis; Bordetella pertussis; Brucella; Legionella pneumophia;Vibrio cholera; Moraxella catharralis; Pseudomonas aeruginosa; Yersinia;aid Kiebsiella pneumonia.

As mentioned in the introduction, detoxified LPS may be useful asvaccinal agent against Gram-negative bacteria infection.

Meningitis is a life-threatening disease of either viral or bacterialorigin. H. influenzae and N. meningitidis are respectively responsiblefor about 40 and 50% of bacterial meningitis. While a vaccine against H.influenzae has been on the market for more than 10 years, there is stilla need for a vaccine against N. meningitidis.

Meningococcal invasive diseases may manifest as either an inflammationof the meninges of the brain and spinal cord (meningitis) or a systemicinfection of the blood (meningococcal sepsis or meningoccaemia).

Meningococci are classified using serological methods based on thestructure of the polysaccharide capsule. Thirteen antigenically andchemically distinct polysaccharides capsules have been described. Almostall the invasive meningococcal diseases are caused by five serogroups:A, B, C, Y and W-135. The relative importance of each serogroup dependson the geographic location. Serogroup B is responsible for the majorityof meningococcal diseases in temperate countries.

While conjugated polysaccharide vaccines already exist against serogroupA, C, Y and W-135, there is currently no vaccine available against theserogroup that is prevalent in the USA and Europe. Indeed, the use ofcapsular polysaccharide as a vaccinal agent for preventing menB diseaseshas been problematic.

Therefore, the use of N. meningitidis LPS as vaccinal agent, in a fullyantigenic and ad hoc detoxified form, is a promising alternative thatmay offer a desirable vaccinal coverage, in particular to serogroup B.

As mentioned hereinabove in the introduction, the major constituent ofthe cell wall of Gram-negative, non-enteric bacteria such as Neisserias,Bordetellas, Haemophilus and Moraxellas, is a lipooligosaccharide (LOS)rather than a true LPS. Nevertheless, for the purpose of thisapplication, the term LPS shall be understood as encompassing LOS. LOSsconstitute a particular sub-class of LPS. The terms “meningococcal LPS”and “meningococcal LOS” are used hereinafter interchangeably.

FIG. 1 shows a scheme of the structure of a N. meningitidis LOS. LOS isconstituted by a branched oligosaccharide composed of 5 to 10monosaccharides linked to lipid A by a KDO. Lipid A and the inner coreconstituted by two KDO, two heptoses (Hep I and II) and a N-acetylatedglucosamine (GlcNAc), are conserved intraspecies. The remaining of theoligosaccharide chains that constitutes the outer core α-chain attachedto HepI; β-chain attached to position 3 of HepII; and γ-chain attachedto position 2 of HepII) is variable according to the immunotypes (ITs).

N. meningitidis LPS can be classified into 13 immunotypes, based ontheir reactivity with a series of monoclonal antibodies (Achtman et al,1992, J Infect. Dis. 165: 53-68). Differences between immunotypes comefrom variation in the composition and conformation of theoligosaccharides chains. This is to be seen in the table hereinafter.

Additional HepII substituents in IT α-chain β-chain position 6 or 7γ-chain L1 NeuNAcα2-6Galα1-4Galβ1-4Glcβ1-4 PEA (1-3) None GlcNAcα1-2 L2NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4 Glcβ1-4 Glcα (1-3) PEA (1-6) ou(Ac_(0.4))-GlcNAcα1-2 PEA (1-7) L3 NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-4 PEA (1-3) None GlcNAcα1-2 L4 NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-4 H (3) PEA (1-6) Ac_(0.5)-GlcNAcα1-2 L5NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-4Glcβ1-4 Glcα (1-3) None(Ac_(0.6-0.4))-GlcNAcα1-2 L6 GlcNAcβ1-3Galβ1-4 Glcβ1-4 H (3) PEA (1-6)ou GlcNAcα1-2 PEA (1-7) L7 NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4 Glcβ1-4PEA (1-3) None GlcNAcα1-2 L8 Galβ1-4 Glcβ1-4 PEA (1-3) None GlcNAcα1-2L9 Galβ1-4GlcNAcβ1-3 Galβ1-4 Glcβ1-4 PEA (n.e.) n.e. GlcNAcα1-2 L10Galβ1-4GlcNAcβ1-3Galβ1-4 Glcβ1-4 PEA (n.e.) n.e. (n.e.)-GlcNAcα1-2 L11Galα1-4Galβ1-4Glcβ1-4 PEA (n.e.) n.e. (n.e.)-GlcNAcα1-2 L12 n.e. PEA(n.e.) n.e. (n.e.)-GlcNAcα1-2 L13 n.e. n.e. n.e. n.e.

As indicated in the above table, a phospho ethanol amine (PEA) replacesthe Glc of the β-chain at position 3 of HepII in LOS L1, L3, L7 and L8.A PEA is attached in position 6 or 7 in LOS L2, L4 and L6. LOS L2, L3,L4, L5, L5, L7 may also be sialylated with N-acetyl neuraminidic acid,on the terminal galactose (Gal) of the α-chain.

Immunotypes L1-L8 are essentially associated with serogroups B and C,while immuno-types L9-L12 are found predominantly within serogroup A.

While any LOS can be equally detoxified, it may be advantageous toemploy LOS L8 in the complexes of the invention as these latter arefurther intended to vaccinal use. Indeed, the complete structure of theLOS L8 α-chain is common to all the immunotypes for which the structurehas been identified so far (Kahler & Stephens, 1988, Crit. Rev.Microbiol. 24: 281).

Meningococcal strains frequently express several immunotypes, thepresence of which may be influenced by the culture conditions. If thereis a special interest in LOS L8, it may be desirable to extract this LOSfrom a strain known to predominantly express the L8 immunotype, or evenbetter, to exclusively express it. Strain A1 (also called 2E) ofserogroup A, strain M978 of serogroup B (Mandrell & Zollinger, 1977,Infect. Immmun. 16: 471; Gu et al, 1992, J. Clin. Microbiol. 30:2047-2053; Zhu et al, 2001, FEMS Microbiol. Lett. 203: 173), strain 8680of serogroup B (Dominique Caugeant collection) and strain 8532 (U.S.Pat. No. 6,476,201) are suitable to this end. These strains areobtainable from the scientific community (U.S. Pat. No. 6,531,131).

Monoclonals that are specific for LOS L8 include Mab 2-1-18 (Moran etal, 1994 Infect Immun. 62: 5290-5295; Mandrell et al, 1986, InfectImmun. 54: 63-69) Mab 6E7-10 (Braun et al, 2004, Vaccine 22: 898-908)Mab 4387A5 and 4385G7 (Andersen et al, 1995, Microb. Pathog. 19:159-168; Gu et al (supra)).

For use in the complexes of the invention, LPS may be obtained byconventional means; in particular it may be extracted from aGram-negative bacterial culture and then purified according to classicalprocedures. Numerous descriptions of such procedures may be found in theliterature. This includes i.a. Gu & Tsaï, 1993, Infect. Immun. 61 (5):1873, Wu et al, 1987, Anal. Biochem. 160: 281 and U.S. Pat. No.6,531,131 all cited by way of illustration only. An LPS preparation mayalso be quantified according to procedures well-known in the art. Aconvenient method is the KDO dosage with high performance anion exchangechromatography (HPAEC) PAD.

LPS may be complexed to the compounds of the invention as such or in aconjugated form. LPS conjugates can be conventionally prepared bycovalently linking LPS to a carrier molecules, e.g. a polypeptide or apeptide; either through a direct covalent link or using chemicalspacer/linker molecules. Examples of carrier molecules include thepertussis, diphtheria or tetanus toxoid and outer membrane proteins(OMP) such as the OMP1 or OMP2/3 of N. meningitidis. Numerousdescriptions of such conjugation processes may be found in theliterature. U.S. Pat. No. 6,531,131 is cited by way of illustrationonly.

When used in a conjugate form, the LPS is advantageously conjugatedbefore being complexed to the compounds of the invention. This beingsaid, non-conjugated LPS is suitable as well.

The invention also relates to:

-   -   A process for detoxifying Gram-negative bacteria LPS, which        comprises mixing together (i) a LPS of Gram-negative bacteria        and (ii) a compound of the invention; and    -   A process for preparing a LPS-peptide complex, which comprises        mixing together (i) a LPS of Gram-negative bacteria and (ii) a        compound of the invention.

For use in the processes of the invention, both constituents areadvantageously in a liquid medium, suitably water. LPS and compoundsolutions are advantageously sterilized before mixing. The preparationprocess is advantageously achieved under sterile conditions. Uponmixing, a precipitate containing the complex is formed. It can berecovered i.a. by centrifugation, and submitted to one or severalwashing steps, if necessary.

As mentioned above, LPS-peptides complexes of the invention are usefulin that they can be safely administered to mammals. Indeed, LPS isdetoxified to such an extent that adverse events shall not occur uponadministration. As a matter of example, a LPS-peptide complex thatexhibits a pyrogenic threshold superior to 1, preferably 10 ng/mL/kg IVdose in the rabbit pyrogen assay, is suitable. Alternatively oradditionally, one may refer to the LAL assay. As vaccines containing LPSamounting 3,000-5,000 LAL endotoxin units have already been authorizedfor human administration (Frederiksen et al, 1991, NIPH Annals 14 (2):67), it is possible to predict that a dose of the vaccine of theinvention may safely exhibit 5,000 LAL endotoxin units or less, e.g.less than 3,000, 2,000, 1,000 or 500 LAL endotoxin units.

As a matter of example, a complex that exhibits e.g. 100 endotoxin units(EU)/μg in the LAL assay, may be therefore acceptable for administrationat a dose of 20 μg. This is achievable with the complexes of theinvention as they may exhibit an LAL activity inferior to 50 EU/μg,frequently inferior to 20 EU/μg.

Further, LPS-peptides complexes of the invention are stable, even inphysiological conditions. By “stable” it is meant that thedetoxification status of LPS in the complexes remains constant overtime, at least 3, 6, 12 or 18 months. This can be monitored byevaluating the detoxification ratio at intervals, i.e. in at least oneof the assays listed above. No significant difference is observed in thedetoxification ratio over time.

LPS-peptides complexes of the invention are also useful in that they areable to induce an immune response against Gram-negative bacteria. Thismay be shown upon administration of complexes to mammals, e.g. rabbits,mice or humans, followed by ELISA analysis of the sera to reveal thepresence of antibodies (i.a. immunoglobulins G or M) specific for LPS.Advantageously, the immune response (antibodies induced) may havebactericidal and/or opsonic activity.

The ability of the immune response induced by the complexes of theinvention to protect against Gram-negative bacteria infection may beevaluated in appropriate animal models that are currently specific for abacterial species or disease. It is within the skills of theprofessionals in the art of vaccines to select a known animal model withregard to a particular bacteria or disease.

As a matter of example, the ability of the immune response induced bythe complexes of the invention to protect against N. meningitidis may beevaluated in the mouse intraperitoneal infection model (Schryvers et al,1989, Infect. Immun. 57 (8): 2425 and Danve et al, 1993, Vaccine 11(12): 1214). It may be also evaluated in humans by measuring thebactericidal activity of the human serum after a complex isadministered. Indeed, this test has been proposed to serve as asurrogate test of protection at least for N. meningitidis serogroup B(Holst et al, 2003, Vaccine, 21: 734). A human serum bactericidalactivity (SBA) titer superior or equal to 4 has been shown to correlatewith protection.

In view of this, the invention also relates to:

-   (i) The use of a LPS-peptide complex of the invention, for treating    or preventing a Gram-negative bacterial infection;-   (ii) A pharmaceutical (vaccinal) composition comprising a    LPS-peptide complex of the invention and a pharmaceutically    acceptable diluent or carrier;-   (iii) The use of a LPS-peptide complex of the invention, in the    preparation of a medicament for treating or preventing a    Gram-negative bacterial infection;-   (iv) A method for inducing an immune response in a mammal against a    Gram-negative bacteria LPS or a Gram-negative bacteria, which    comprises administering an effective amount of a LPS-peptide complex    of the invention, to the mammal; and-   (v) A method for treating or preventing a Gram-negative bacterial    infection, which comprises administering a therapeutically effective    amount of a LPS-peptide complex of the invention, to an individual    in need.

A vaccinal composition of the invention can be administered by anyconventional route, in particular by systemic or intramuscular route; asa single dose or as a dose repeated once or several times, e.g. two orthree times at intervals, e.g. at 1, 2, 3, 6, 10, 12 month-interval. Avaccinal composition of the invention can be conventionally formulated,advantageously in liquid form. If necessary, an adjuvant can be added tothe vaccinal composition of the invention; however, it is indicated thatcomplexes of the invention can be sufficiently immunogenic so that thepresence of adjuvant in the vaccinal compositions is not required.

The appropriate dosage depends on various parameters, for example theindividual treated (adult or child), the mode and frequency ofadministration and the LPS detoxification status, as can be determinedby persons skilled in the art. In general, it is indicated that a dosefor administration to a human adult should not excess 10,000;advantageously 8,000; preferably 5,000; more preferably 1,000; mostpreferably 500 LAL Endotoxin Unit. In the LAL assay, the value measuredfor a complex of the invention may commonly be as low as 10-20 EU/μg.Therefore, a dose can contain from 1 to 500, advantageously from 2.5 to100, preferably from 10 to 50, more preferably from 15 to 30 μg.

It is reminded that, by convention, amounts of complex are alwaysexpressed as LPS content. Accordingly and by way of example only, “50 μgof complex” actually means 50 μg of LPS in the complex preparation.

The Examples reported hereinafter further illustrate the invention byreference to the following figures.

FIG. 1A shows the structure of the LPS L8 of N. meningitidis. Kdo standsfor 2-keto, 3-desoxy octulosonic acid; Hep stands for heptose; Glcstands for glucose; Gal stands for galactose; and GlcNAc stands forN-acetylated glucosamine.

FIG. 1B shows the reaction that occurs upon LPS treatment with aceticacid.

FIGS. 2A-2C show the HPLC chromatogram obtained at 214 nm with acomposition essentially comprising the SAEP2-L2 peptide in monomericform (2A), in parallel dimeric form (2B) and anti-parallel dimeric form(2C). Coordinates are: times (min) and absorbance unit (AU).

FIG. 3 shows the HPLC chromatogram obtained at 214 nm with a compositioncomprising the SAEP2-L2 peptide in monomeric form, parallel dimeric formand anti-parallel dimeric form.

FIGS. 4A-4C show the ¹H NMR spectra obtained with a compositionessentially comprising the SAEP2-L2 peptide in monomeric form (4A), inparallel dimeric form (4B) and anti-parallel dimeric form (3C). In allof them, a peak at 1.9 ppm indicates that the peptide is in an acetatesalt form.

FIGS. 5A-5C show an enlargement of the region of the ¹H NMR spectra ofFIGS. 4A-4C comprised between 6.5 and 7.5 ppm.

FIG. 6 shows the 6.5-7.5 ppm region of the ¹H NMR spectrum obtained witha composition comprising the SAEP2-L2 peptide in monomeric form,parallel dimeric form and anti-parallel dimeric form.

FIGS. 7A-7C show the MALDI-ToF spectra of the calibration standard (7A),the parallel dimer (7B) and the anti-parallel dimer (7C).

FIG. 8 shows the HPEAC-PAD chromatogram of LPS hydrolysed by acetic acidtreatment.

EXAMPLE 1 Preparation of the SAEP2-L2 Parallel Dimer 1.1. Synthesis

The synthesis of the corresponding linear monomer is achieved on solidphase using a computer-driven automatic synthesizer Milligen 9050(Millipore Inc.) operating with columns containing resin supports e.g.polyoxyethylene glycol-activated polystyrene, or activatedpolyacrylamide, which are appropriately activated according to thechoice of the first amino acid of the selected peptide sequence asreported by Atheron & Shepard: in Solid phase peptide synthesis, 1989,IRL press, Oxford U.

The synthesis cycle proceeds step-by-step, according to the reportedlinear sequence. It is performed in pure solvent dimethylformamide (DM).Side-protected, activated amino acids are used.

The thiol group of the Cys residue in position 10 (Cys-10) is protectedwith the acid-labile group Trityl (triphenyl-methyl derivative, Trt).The thiol group of the Cys residue in position 4 (Cys-10) is protectedwith the acid-resistant group S-acetamido-methyl (Acm).

All the amino acids are activated at the —COOH side byO-penta-fluorophenyl-phosphate esters (O-Pfp-derivatives). They aretemporarily protected at the —NH₂ side by 9-fluorenyl-methyloxy-carbonylesters (Fmoc-derivatives).

Once synthesized, the protected peptide is cleaved from the resinsupport using TFA 95% in the presence of the scavenger ethandithiol at2-5% (v/v). In these conditions, the thiol group of the Cys-10 isde-protected while the thiol group of the Cys-4 remains Acm-protected.The free, Acm-protected peptide is concentrated by vacuum-evaporationand then recovered by precipitation with ether at 80% (v/v) finalconcentration.

The Cys-4 protected, Cys-10 de-protected peptide is dried under vacuum,then solubilized in water at the concentration of 1 to 10 mg/mL andadjusted at pH 7.50 with 0.1 M aqueous ammonia. In order to achievedimerization through the Cys 10 residues, oxidation is then performed byvigorous stirring of the aqueous solution at 4° C., under a pressure of1 Atm, for 18-24 hours. Complete oxidation of the thiol groups isdetermined by the Elman colorimetric assay.

The partly oxidized peptide in solution at the concentration of 1 to 10mg/mL is then processed for de-protection of the remaining Cys-4 S-Acmfunctions. To this end, the peptide solution is added with mercuricacetate at a final concentration of 0.1 M, using phenol at 2-5% (v/v) asscavenger. The solution is again vigorously stirred at 20° C., under apressure of 1 Atm, for 18-24 hours. Complete oxidation of the thiolgroups is determined by the Elman colorimetric assay.

1.2. Purification

In order to remove the low-MW molecules contained in the peptidepreparation (scavenger, mercuric acetate etc.), this latter is appliedon a reverse-phase column Sep-Pack (Millipore) operated under pressureof 1 Atm. In an aqueous solvent, the peptide is retained on the columnby hydrophobic forces, while all the hydro-soluble, low-MW molecules gowith the flow-through. The peptide is then eluted by a mixture ofmethanol-water 50-70% (v/v). The peptide eluted in the alcoholicsolvent, is recovered by vacuum concentration and solubilized again inwater at the desired concentration.

Final purification is achieved on HPLC-operated reverse-phase C18 column(dimensions=250×4 mm) using a linear gradient 0-100% of Solvent A (0.1%TFA (trifluoroacetic acid) in water) and Solvent B (nitryl acetate 80%in water). In these conditions, the parallel dimer elutes as a singlesharp peak. Peak fractions are recovered.

The preparation is kept in lyophilized form, at +2-+6° C., under aneutral gas, argon or nitrogen.

1.3. Characterization of the Purified Peptide 1.3.1. Amino AcidComposition

The amino acid composition is analysed by the Pico-Tag method(Millipore). Results are reported in the table hereinafter.

Amino acid Theoretical (moles/mole) Found (moles/mole) Lysine 6.0 5.90Threonine 2.0 2.00 Phenylalanine 2.0 2.05 Leucine 6.0 6.10 Cysteine 4.03.85

1.3.2. Molecular Mass

The molecular mass is measured by Ion Cyclotron Resonance (ICR). Thevalue found is 2,387.33±0.3 AMU, a value coherent with the elementarystructure C₁₁₀H₁₉₀O₂₄N₂₆S₄ of the peptide formula.

EXAMPLE 2 Preparation of the SAEP2-L2 Monomer and Anti Parallel Dimer2.1. Synthesis

The synthesis of the linear monomer is performed as in Example 1, exceptthat a the different methodology is used for protecting the thiol groupsof the cysteine residues: Both Cys-4 and -10 are protected at their —SHgroup by the acid-labile group Trityl (triphenil-methyl, Trt).

The protected peptide is cleaved from the resin support by TFA 95%, inthe presence of the scavenger Ethandithiol at 2-5% (v/v). In theseconditions, the thiol groups of both Cys-4 and 10 residues arede-protected. The cleaved and de-protected peptide is then concentratedunder vacuum-evaporation and recovered by precipitation with ether 80%(v/v).

The de-protected peptide is solubilized in water at the concentration 1to 10 mg/mL and the pH is adjusted to 7.50 with 0.1 M aqueous ammonia.

Oxidation is then performed by vigorous stirring of the aqueous solutionfor 18-24 hours, at 4° C., under pressure of 1 Atm. Complete oxidationof the thiol groups is determined by the Elman colorimetric assay.

2.2. Purification of the Peptides

The peptides in solution actually constitute a mixture of cyclic monomer(about 40%) and anti-parallel dimer (about 60%). Each form is purifiedby preparative Reverse-phase HPLC chromatography. Indeed, it is possibleto separate the cyclic monomer from the anti-parallel dimer since theseforms elute, each as a single sharp peak, at different retention times.The anti-parallel dimer elutes at a lower retention time. This isconsistent with the different molecular symmetry of the two dimers. Theanti-parallel peptide may assume a lower minimal energy in aqueoussolvents by virtue of its lower internal steric hindrance of theside-chains, similarly to the “trans” vs “cis” conformation of any otherisomeric entities.

All preparations are kept in lyophilized form, at +2-+6° C., under aneutral gas, argon or nitrogen.

2.3. Characterization of the Antiparallel Dimer 2.3.1. Amino AcidComposition

The amino acid composition is analysed by the Pico-Tag method(Millipore). Results are reported in the table hereinafter.

Amino acid Theoretical (moles/mole) Found (moles/mole) Lysine 6.0 6.10Threonine 2.0 1.95 Phenylalanine 2.0 1.90 Leucine 6.0 6.05 Cysteine 4.03.90

2.3.2. Molecular Mass

The molecular mass is measured by Ion Cyclotron Resonance (ICR). Thevalue found is 2,387.30±0.3 AMU, a value coherent with the elementarystructure C₁₁₀H₁₉₀O₂₄N₂₆S₄ of the peptide formula.

EXAMPLE 3 Further Characterization of the Monomer, Parallel andAntiparallel Dimers by HPLC-Reverse Phase, NMR and MALDI-ToF MassSpectrometry

The dimeric parallel peptide as prepared in Example 1 and the monomericand dimeric antiparallel peptides as prepared in Example 2 arecharacterized by HPLC-reverse phase (FIGS. 2A-2C) and NMR (FIGS. 4A-4Cand 5A-5C).

3.1. Characterization by HPLC-Reverse Phase Experimental Conditions

This technique is carried out on a HPLC chain (Waters™), using theMillennium software 32 V30501 (Waters™) for data acquisition. Theanalytical column Macherey Nagel™ ref 720014.6 (Nucleosil 5 μm C18 100Angström 250×4.6 mm) is operated at 25° C.

30-40 μg of each lyophilised peptide are diluted first in 30 μl water;to which is added 30 μl of trifluoroacetic acid (TFA) 0.1% in water.

A mixture of the monomeric, dimeric parallel and antiparallel peptidesis also prepared by mixing 40 μg of a powdered preparation of eachpeptide in 60 μl water; to which is added 60 μl of TFA 0.1% in water.

The column is equilibrated using 20% phase mobile B (TFA 0.1%, CH₃CN 80%in water). Once samples are applied to the equilibrated column, thephase B gradient runs from 20 to 60% within 40 min (1% B/min), at a flowrate of 1 mL/min.

Detection is achieved at 214 nm. Results are to be seen in FIGS. 2A-2C.

Results

Each peptide is eluted at a different retention time In the experimentalconditions described above, elution occurs at the following retentiontime (RT):

-   -   monomer: RT=28.283 min    -   parallel diner: RT=29.708 min    -   antiparallel dimer: RT=22.059 min

The HLC-RP technique is used to verify the purity of each peptidepreparation. The relative purity degree of each peptide is calculated byintegrating the peak surfaces. It is expressed as the peptide peaksurface/surfaces of the whole peaks.

In FIG. 2A-2C, it can be seen that the monomer and parallel andantiparallel dimer preparations exhibit a purity degree of 98, 96.9 and97% respectively.

FIG. 3 shows the HPLC chromatogram of the mixture.

3.2. Characterization by NMR Experimental Conditions

¹H NMR analysis (500 MHz, 25° C., HOD presaturation) is carried outusing samples of peptides diluted in a H₂O/D₂O mixture (90/10 v/v). ABruker™ DRX500 spectrometer and associated software for data acquisitionare used.

In more details, peptides preparation kept at −70° C. are used foranalysis. Dimeric peptide solutions 0.5 mM are prepared while diluting1.33 g in 1 mL H₂O. 144 μl of the solutions are mixed with 16 μl of D₂O99.9% D in 3 mm NMR tubes. For calibration, an external solution ofTSP-d4 (3-(trimethylsilyl)propionic-2,2,3,3,-d4 acid sodium salt;Aldrich ref 29304-0) 0.075% (w/w) in H₂O/D₂O mixture (90/10 v/v) isused. The spectrometer is calibrated so that the unique resonance signalof TSP-d4 be at 0 ppm.

Results

In the experimental conditions used, ¹H NMR spectra of the monomer anddimers cover a range from 0 to 9.5 ppm and are composed of 3 mainregions:

-   -   from 6.5 to 7.5 ppm;    -   from 5.5 to 2.5 ppm; and    -   from 2 to 0.3 ppm.        This is to be seen in FIG. 4A-4C.

¹H NMR spectrum of the monomer is characterized by a NMR pattern of 5aromatic protons that are expected between 7.25 and 7.45 ppm, in theexperimental conditions reported hereinabove. In the experiment reportedin FIG. 5A, this NMR pattern is itself composed of a first multipletfrom 7.25 to 7.35 ppm with an integral curve corresponding to 3H and asecond multiplet (pseudo-triplet), centered at 7.39 ppm with an integralcurve of 2H. This latter signal is characteristic of the monomer only.

¹H NMR spectrum of the parallel dimer is characterized by a doubletsignal between 7.10 and 7.25 ppm corresponding to 4 aromatic protons anda multiplet between 7.25 and 7.40 ppm with an integral curve of 6H. Inthe experiment reported in FIG. 5B, the 4H doublet is found centered at7.185 ppm (pics at 7.18 and 7.19 ppm).

¹H NMR spectrum of the antiparallel dimer is characterized by a doubletsignal 4 aromatic protons between 6.95 and 7.10 ppm and a multipletbetween 7.10 and 7.30 ppm with an integral curve of 6H. In theexperiment reported in FIG. 5C, the 4H doublet is found centered at7.025 ppm (pics at 7.02 and 7.03 ppm).

As shown in FIG. 4C, the ¹H NMR spectrum of the antiparallel dimer isalso characterized by two upfield methylic resonances that are expectedbetween (i) 0.40 and 0.65 (doublet) and (ii) 0.70 and 0.85 ppm(doublet). In one experiment, these doublets are found centered at 0.42and 0.68 ppm. They are observed neither in the monomer, nor in theparallel dimer.

3.3. Identification by MALDI-ToF Mass Spectrometry

Analysis by MALDI-ToF (Mass Assisted Laser Desorption Ionisation-Time ofFlight) mass spectrometry allows determining the monoisotopic mass ofthe peptide. This technique does not discriminate the antiparallel andparallel dimers.

Experimental Conditions

MALDI-ToF analysis is achieved using the Biflex III mass spectrometer(Bruker™) and associated softwares, in a positive reflector mode.Peptides are mixed with a matrix (alpha cyano-4hydroxy cinnamic acid)that absorbs laser energy.

The spectrometer is externally calibrated with a mixture of syntheticpeptides (ACTH 18-39 (adenocorticotropic fragment 18-39) bombesine, andsomatostatine 28.

A saturated HCCA matrix solution is prepared while diluting 50 mg HCCAin 300 μl 70% ACN (acetonitril) 0.1% TFA (trifluoroacetic acid) inwater.

A ½ saturated HCCA solution is further prepared while diluting vol:volwith 30% ACN, 0.1% TFA in water.

For calibration, primary standard solutions are first prepared in 0.1%TFA. They are as follows:

-   -   Adenocorticotropic fragment 18-39 (ACTH 18-39): 100 pmoles/μl        (0.247 mg/mL);    -   Bombesine: 100 pmoles/μl (0.160 mg/mL); and    -   Somatostatine 28: 100 pmoles/μl (0.31 mg/mL).

A secondary standard solution is prepared as follows:

ACTH 100 pmoles/μl 2 μl Bombesine 100 pmoles/μl 4 μl Somatostatine 100pmoles/μl 4 μl ACN 30%, TFA 0.1% 50 μl 

Peptide solutions at 1 mg/mL in water are diluted down to 0.02 mg/mLwith 30% ACN, 0.1% TFA in water.

Calibration and peptide samples are diluted vol:vol with the ½ saturatedHCCA solution. Droplets of about 1 μl are deposited on a steel target(Bruker™) and dried by evaporation.

Results

Results are to be seen in FIGS. 7A-7C.

The theoretical monoisotopic masses calculated by the software based onthe amino acid sequences are:

ACTH 28 M+H⁺=2465.199 Da Bombesine M+H⁺=1619.823 Da Somatostatine 28M+H⁺=3147.471 Da SAEP2-L2 M+H⁺=2388.35 Da.

La norme retenue pour le contrôle est fixée à±2 Da par rapport à la massthéorique.

As shown in FIG. 7A, the experimental values found for the calibrationpeptides are 2465.225, 1619.814 and 3147.454 Da respectively. L'ecart demesure interne est donc (0.026+0.009+0.017)/7232.493=7.2 ppm.(authorized <50 parts per million).

As shown in FIGS. 7B and 7C, the experimental values found for theparallel and antiparallel dimer preparations are 2388.449 and 2388.532Da. These values are within the identity range (+2 Da) centered on thetheoretical values range. This means that the samples contain what isexpected.

EXAMPLE 4 Preparation of a LPS L8/Peptide I″ Complex/Aggregate 4.1.Preparation of LPS L8 4.1.1. Meninge Culture

Preculture: Two mL frozen samples of working seed from a N. meningitidisA strain known to express LPS exclusively under the L8 form, are used toinoculate in a 2 l erlen containing 200 mL of Mueller-Hinton broth(Merck) complemented with 4 mL of a glucose solution in water (500g/l).This operation is repeated 4 times. Erlens are incubated at 36±1° C. for10±1 hrs while stirring (100 rpm).

Culture: The erlen contents are gathered together and the preculture iscomplemented with 400 mL of a glucose solution in water (500 g/l) and800 mL of an amino acid solution. This preparation is used to inoculatethe Mueller-Hinton broth, in a 30 l fermentor (B. Braun™) at an initialOD_(600nm) close to 0.05. Fermentation is performed overnight at 36° C.,pH 6.8±0.2, 100 rpm, pO₂ 30%, and initial flow rate of the air 0.75l/min/L culture. After 7±1 hrs, (OD_(600nm) about to 3), the culture isfeeded by MH broth at a flow rate of 440 g/h. When the glucoseconcentration is lower than 5 g/l, the fermentation is stopped. Usually,the final OD_(600nm) is comprised between 20 and 40. Cells are collectedby centrifugation for 1 h 30 at 7000 g at 4° C. Pellets are kept frozenat −35° C.

4.1.2. Purification of LPS First Phenol Extraction

Pellets are defrosted and suspended with 3-volume phenol 4.5% (v/v) andstirred vigorously for 4 hrs minimum at about 5° C.

The bacterial suspension is heated at 65° C. and then mixed v/v withphenol 90% at 65° C. The suspension is stirred vigorously, at 65° C. for50-70 min and then cooled down to about 20° C.

The suspension is centrifuged for 20 min, at 11 000 g, at about 20° C.The aqueous phase is collected and kept. The phenol phase and theinterphase are recovered and submitted to a second extraction.

Second Phenol Extraction

The phenol phase and the interphase are heated at 65° C. and mixed witha volume of water equivalent to the volume of the aqueous phase that waspreviously collected. The mixture is stirred vigorously for 50-70 min at65° C. and then cooled down to about 20° C. The mixture is centrifugedfor 20 min, at 11 000 g, at about 20° C. The aqueous phase is collectedand kept. The phenol phase and the interphase are recovered andsubmitted to a third extraction.

Third Phenol Extraction

Procedure for the second extraction is repeated.

Dialysis

The 3 aqueous phases are dialysed overnight and separately against 40 lof water. The dialysates are pooled together. The dialysate pool isadjusted with Tris 20 mM, MgCl₂ 2 mM (one volume per 9 volumes of thedialysate pool). pH is adjusted to 8.0±0.2 with NaOH 4 N.

DNAse Treatment

250 UI of DNAse is added per gram of treated bacterial pellet (wetweight). The preparation is stirred at 37±2° C. for 55-65 min. pH isadjusted at 6.8±0.2. The preparation is filtered on 0.22 μm membranes.

Gel filtration: The preparation is purified on a Sephacryl S-300 column(5.0×90 cm; Pharmacia™).

First Alcoholic Precipitation

Powder of MgCl₂, 6H₂O is added to the LPS-containing fractions pooledtogether, to reach an MgCl₂ concentration of 0.5 M and dissolved whilestirring.

While stirring at 5±3° C., dehydrated absolute alcohol is added to afinal concentration of 55% (v/v). Stirring is performed overnight at5±3° C., followed by centrifugation at 5,000 g for 30 min at 5±3° C. Thesupernatants are discarded and the pellets are submitted to a secondextraction.

Second Alcoholic Precipitation

The pellets are resuspended with at least 100 mL MgCl₂ 0.5 M, whilestirring. The previous procedure is repeated. Pellets are resuspendedwith at least 150 mL water.

Final step: Gel filtration is repeated and the LPS-containing fractionspooled together are finally sterilised by filtration (0.8-0.22 μm) andkept at 5+3° C.

As a preliminary control, the LPS preparation is analyzed by SDS-PAGEelectrophoresis. Upon silver nitrate staining, a single large band isrevealed This indicates at least that the preparation does not containany entity other than LPS L8.

The purification process as described allows obtaining about 150 mg LPSL8 per culture liter (yield about 50%).

4.1.3. LPS L8 Quantification: KDO Dosage with HPAEC-PAD

The bibliographic reference for this technique is Kiang et al, (1997)Determination of 2-keto-3-deoxyoctulosonic acid (KDO) with highperformance anion exchange chromatography (HPAEC): Survey of stabilityof KDO and optimal hydrolytic conditions Anal. Biochem. 245: 7.

As shown in FIGS. 1A-1B, LPS comprises in its structure 2 KDO units, onebeing in a lateral position.

LPS quantification is achieved through dosage of the lateral KDO unitliberated upon soft acid hydrolysis (See FIG. 1B).

Acid Hydrolysis

Samples of the LPS preparation obtained after the last diafiltration ofsection 4.1.2. are recovered and diluted with water under a final volumeof 400 μl in Dionex™ 1.5 mL flasks so that LPS concentration of thesamples falls under the etalon range (1.4-72.1 μg/mL).

Samples to be quantified as well as the KDO etalon range are proceededas follows 100 μl of the hydrolysis solution (acetic acid 5%; glucuronicacid (GlcA) 20 μg/mL) are added. Hydrolysis is performed for 1 h at 100°C. Flasks are then dried at 40° C. under nitrogen and filled with 400 μlwater.

Dosage

This technique is carried out on a HPAEC chain (Dionex™), using theChromeleon Dionex™ software for data acquisition. The analytical columnCarbopac PA1 4×250 mm (Dionex™) is operated at 30° C.

The column is equilibrated with the elution solution (NaOH 75 mM, AcONa90 mM). 100 μl of sample are injected into the column. Then the columnis submitted to an elution flow rate of 1 mL/min for 22 min.

Chromatogram of LPS sample is to be seen in FIG. 8. The KDO amountpresent in the sample is determined by integration of the KDO peak. Asone KDO mole liberated by hydrolysis corresponds to one LPS mole, it ispossible to determine the LPS concentration of the initial preparation.

4.2. Preparation of Peptides

Peptides are prepared according to the processes described in Examples 1and 2 above.

4.3. Preparation of the LPS L8/Peptide I″ Complex/Aggregate

Purified LPS is used as pseudo-solution at 1 mg/mL in sterile, pyrogenfree water (Milli Q quality, adjusted to pH 7.2 Limulus negative). Thetranslucid pseudo-solution is sterilized by filtration using a 0.22 μmmembrane.

A solution of peptide SAEP2-L2 at 1 mg/mL in sterile, pyrogen-free water(Milli Q quality, adjusted to pH 7.2, Limulus negative) is alsosterilized by filtration on 0.22 μm membrane.

All the next steps are achieved under sterile conditions.

One volume of the LPS pseudo-solution is added to one volume of thesolution of peptide SAEP2-L2. A precipitate (endotoxoid complex)immediately appears. Stirring is carried out for 5 min at roomtemperature. The preparation is left to stand at +4° C. overnight.

The precipitate (Endotoxoid) is then recovered by centrifugation at 3000rpm for 10 min. The supernatant is discarded.

The pellet is washed with one volume of sterile, pyrogen free water(Milli Q quality, adjusted to pH 7.2, Limulus negative).Centrifugation/washing steps are repeated five times.

At last, the pellet is resuspended in sterile, pyrogen free water (milliQ quality) pH=7.2, at about 1 mg/mL concentration, based on the wetweight of the precipitate. The suspension is stored at +4° C. A KDOdosage is achieved to determine the LPS content and the suspension isadjusted to e.g. 0.50 mg/mL of complex (expressed as LPS content).

-   -   The LPS-peptide complex tested in the following examples is the        LPS-antiparallel dimer complex as obtained in section 4.3,        unless otherwise indicated. Therefore, this specific complex is        simply referred to as LPS-peptide complex.    -   In a similar manner, the LPS as obtained in section 4-2. is        simply referred to as LPS.    -   Comparison of LPS and LPS-peptide complex is achieved using the        LPS lot also used for the preparation of the complex.

EXAMPLE 5 Evaluation of the Detoxification of the LPS-Peptide Complex

Several assays are used to evaluate the detoxification.

5.1. Limulus Amebocyte Lysate (LAL) Assay

In this assay, the ability of the SAEP2-L2 anti-parallel and paralleldimers and the SAEP2-L2 cyclic monomer to detoxify LPS is compared. Tothis end, the LPS-peptide complexes involving the parallel dimer or themonomer are prepared exactly as it is reported in Example 4 for theLPS-antiparallel peptide complex.

LAL is a very sensitive test used to detect and quantify endotoxins ofgram-negative bacteria. The test is based on the property of theamoebocyte lysate protein from horseshoe crab (Limulus polyphemus) toinduce coagulation in the presence of endotoxin.

The evaluation of the LPS endotoxin activity is performed by using theend-point-chromogenic technique, in accordance with the EuropeanPharmacopeia [as described in the European Pharmacopeia techniques(Edition 5.0, paragraph 2.6.14)]. To this end, the kit QCL-1000 ref50-647 U (Cambrex-BioWhittaker™) is used (linear zone of the kit: 0.1 to1 UI/mL) as well as a positive control (E. coli endotoxin, 4 10³ EU/mL,Sigma).

Dilution of (i) samples to be tested, (ii) standard and (iii) positivecontrol are achieved with dilution buffer (Cambrex-BioWhittaker™) tocover the respective ranges: 1/10 to 1/10⁵; 0.5 to 0.031 EU/mL and 1/10⁴to 1.8 10⁴.

50 μl of sample, standard and positive control dilutions are dispensedper well of 96 flat-bottom well ELISA plate. Fifty μl of lysate areadded per well. Incubation is pursued for 10 min at 37° C. Then 100 μlof the p-nitroaniline chromogenic substrate are added. Incubation ispursued for 6 min at 37° C. The chromogenic reaction is stopped whileadding 100 μl freezed acetic acid 25% in water. Plate is read byspectrophotometry at 405 nm.

The results are expressed in Endotoxin Unit (EU)/μg of complex. They areshown in the table hereinafter. The detoxification ratio can beestablished by the LPS/LPS-peptide complex ratio and expressed in logunit.

Mean Detoxification ratio Range value Expressed EU/μg EU/μg in log LPS(6 assays)  1-12 10⁴ 25,000 LPS-antiparallel peptide  5-32 12-201,250-2,000 3.5 complex (13 assays) LPS-parallel peptide 30-40 30-40600-800 3 complex LPS-monomer peptide  >2000 >2000 <12.5 <1 complex

As may be seen, the dimeric forms of the SAEP2-L2 peptide are moreeffective in detoxifying LPS than the cyclic monomer form.

5.2. Pyrogen Test in Rabbits

Rabbit is known to be the animal specie with sensitivity to pyrogeniceffects of LPS equivalent to humans. The pyrogen test consists inmeasuring the rise in body temperature evoked in three rabbits by theintravenous (IV) injection of a sterile solution of the substances to beexamined. The test, reading and calculations are performed in accordancewith the European Pharmacopoeia, (Edition 5.0, paragraph 2.6.8). Thetemperature rise is interpreted depending the summed response of thetemperatures: conformity is met when the summed response does not exceed1.15° C. and non-conformity, when the summed response exceeds 2.65° C.In the present case, the pyrogenic threshold is set up below, between1.15° C. and 2.65° C.

As found, the limit pyrogen dose (IV) in rabbit corresponds to 0.025ng/kg (LPS), and 10-25 ng/kg (LPS-peptide complex). These results showthat the LPS-peptide complex is less pyrogen than LPS, when given by theintravenous route. As measured in this test, the detoxification ratio(LPS-peptide complex/LPS) is between 400 and 1,000.

5.3. Acute Toxicity Assay: LD50 in D-Galactosamine Sensitized MiceReferences for this assay include i.a. Galanos et al, 1979, PNAS 76:5939 Baumgartner et al, 1990, J. Exp. Med. 171 (3): 889 and U.S. Pat.No. 6,531,131.

Groups of eight-week old female inbred mice are injected by theintraperitoneal (IP) route (0.5 mL) with escalating doses of LPS orLPS-peptide complex, just after being treated with D-galactosamine (15mg/0.2 mL) by the IP route (the toxicity of LPS is increased of around1,000 fold with the D-galactosamine treatment which renders the modelvery sensitive). The death rate is then followed during four days.

The LD50 observed with the LPS is 3.6 ng/mouse (1.91-6.70 ng/mouse);whereas that observed with the LPS-peptide complex is 1 μg/mouse (0.2-5μg/mouse), indicating that the detoxification ratio (LPS-peptidecomplex/LPS) is about 250 (100-1000).

5.4. Attenuation of the Pro-Inflammatory Effects of LPS when Completedwith Peptide

In order to evaluate to which extent the LPS-peptide complex canattenuate LPS-induced toxic effects, the effect of the LPS-peptidecomplex on the release of pro-inflammatory cytokines is monitored(assessed) in in vitro and in vivo assays.

-   -   In vivo: cytokine (IL6 and TNFα) releases in the sera of mice        immunized either with LPS or LPS-peptide complex are compared by        ELISA. Blood samples are recovered 90 min after SC immunization,        which is the optimal time for the release of those cytokines.        C3H/HeOuJ, TLR4- -/- -, C3H/HeN and CD1 mouse strains are tested        The two first are sensitive neither to LPS nor LPS-peptide        complex. The third and fourth are both found LPS-sensitive. CD1        mice are found more LPS-peptide complex-sensitive than the        others and therefore selected for further experiments retaining        the most severe conditions.    -   In vitro: cytokine (IL6, IL8 and TNFα) releases from human whole        blood cell cultures stimulated for 24 h at 37° C., with        different concentrations of LPS or LPS-peptide complex are        compared.

5.4.1. In Vivo Assay

CD1 mice are administered subcutaneously (SC) (i) either 10 μg of LPS or(ii) 10 μg of LPS-peptide complex. They are bled 90 minutes afterinjection. IL6 and TNFα releases are measured in the sera by ELISA.

ELISA Detection of Cytokine Secretion

ELISAs are carried out using the OptEIA mouse IL6 and TNFα sets(Pharmingen), each including the capture antibody (anti-mouse cytokine),the detection antibody (biotinylated anti-mouse cytokine),avidin-horseradish peroxidase conjugate and the standard (recombinantcytokine), all from Pharmingen.

Anti-mouse IL6 and TNFα antibodies are 1/250 diluted in 0.1 M carbonatebuffer pH 9.5 (Sigma). For each assay, 100 μl of an antibody dilutionare distributed per well of a Maxisorp NUNC 96 flat-bottom well ELISAplate. Plates are incubated overnight at +4° C.

Plates are washed in PBS 0.05% Tween 20. 200 μl of PBS, 0.5% bovineserum albumin (BSA) saturation buffer are then added per well.Incubation is pursued for one hr at room temperature. Plates are washedin PBS 0.05% Tween 20.

Recombinant IL6 or TNFα cytokine dilutions are prepared in the RPMImedium 1% FCS 10%, within the range of (i) 4,000 pg/mL-62.5 pg/mLstandard. 100 μl of each dilution are distributed per well, to establishthe standard curve.

Serum dilutions are prepared in the RPMI medium P.S. glu 1% FCS 10%.Sera of mice injected with LPS are 1/25 and 1/125 diluted. Sera of miceinjected with LPS-peptide complex are 1/5 and 1/25 diluted. 100 μl ofeach dilution are distributed per well.

Incubation is pursued for 2 hrs at room temperature.

Plates are washed in PBS 0.05% Tween 20. Biotinylated anti-mousecytokine antibodies and the enzyme are each 1/250 diluted in PBS 10%fetal calf serum. 100 μl of each dilution are added per well. Incubationis pursued for one hr at room temperature.

Plates are washed in PBS 0.05% Tween 20. 100 μl of tetramethylbenzidine(TMB) substrate (TMB solutions A and B (KPL) mixed vol/vol) aredistributed in wells. Incubation is pursued for 10-30 min at roomtemperature.

The reaction is stopped by adding 100 μl of 1 M H₃PO₄ per well. Platesare read at 450 nm. Results are to be seen in the table hereinafter.

IL6 release TNFα release Product Mean Mean injected to (pg/mL)Detoxification (pg/mL) Detoxification mice n = 6 (log) ratio (log unit)n = 6 (log) ratio (log unit) LPS 4.7 4.1 LPS-peptide 2.2 2.5 <1 >3.1complex Peptide <1 <1

The peptide alone does not induce IL6 or TNFα. The LPS-peptide complexallows for about 100-fold of detoxification (100-fold decrease in IL6secretion).

5.4.2. In Vitro Assay Preparation of the Test Substances

LPS preparation (1 mg/mL) and LPS-peptide complex (500 μg/mL) are eachdiluted in 10 mM Tris, NaCl 150 mM, 0.05% Tween 20, 5% sucrose to aconcentration of 50 μg/mL. They are further diluted in physiologicalsaline to a concentration of 5 μg/mL.

Serial 1/5 dilutions are performed in AIM-V medium (Gibco (Invitrogen))for each test substance down to a concentration of 2.56 10⁻³ pg/mL

Stimulation

Human blood collected on sodium heparin (25,000 U/5 mLsanofi-synthelabo) is diluted 1:4 (vol:vol) in AIM-V medium anddistributed in Micronics™ tubes (400 μl/tube). 100 μl of a dilution ofthe test substances are added. Peptide and buffer controls are tested at1/20 dilution. Tubes are incubated for 24 hrs at 37° C., in a wetatmosphere at 5% CO₂.

Plasma Recovery

Tubes are then centrifuged for 10 min at 500 g. At least 200 μl ofsupernatant are recovered from each tube and kept frozen at −80° C.until titration.

ELISA Detection of Cytokine Secretion

ELISAs are carried out using the OptEIA human IL6, IL8 and TNFα setsfrom Pharmingen, each including the capture antibody (mouse anti humancytokine), the detection antibody (biotinylated mouse anti-humancytokine), avidin-horseradish peroxidase conjugate and the standard(recombinant cytokine).

Anti-human IL6, IL8 and TNFα antibodies are 1/250 diluted in 0.1 Mcarbonate buffer pH 9.5 (Sigma). For each assay, 100 μl of an antibodydilution are distributed per well of a Maxisorp NUNC 96 flat-bottom wellELISA plate. Plates are incubated overnight at +4° C.

Plates are washed in PBS 0.05% Tween 20. 200 μl of PBS, 0.5% bovineserum albumin (BSA) saturation buffer are then added per well.Incubation is pursued for one hr at room temperature. Plates are washedin PBS 0.05% Tween 20.

Recombinant IL6, IL8 or TNFα cytokine dilutions are prepared in AIM-Vmedium within respective range of (i) 1,200 pg/mL-18.75 pg/mL; (ii) 800pg/mL-12.5 pg/mL; and (iii) 1,000 pg/mL-15.87 pg/mL standard. 100 μl ofeach dilution are distributed per well, to establish the standard curve.

Plasma dilutions are prepared in the AIM-V. Plasmas recovered from bloodstimulated with LPS are 1/25 and 1/125 diluted. Those recovered fromblood in contact with the LPS-peptide complex are 1/5 and 1/25 diluted.100 μl of each dilution are distributed per well.

Incubation is pursued for 2 hrs at room temperature.

Plates are washed in PBS 0.05% Tween 20. Biotinylated anti-humancytokine antibodies and the enzyme are each 1/250 diluted in PBS 10%fetal calf serum. 100 μl of each dilution are added per well. Incubationis pursued for one hr at room temperature.

Plates are washed in PBS 0.05% Tween 20. 100 μl of tetramethylbenzidine(TMB) substrate (TMB solutions A and B (KPL) mixed vol/vol) aredistributed in wells. Incubation is pursued for 10-30 min at roomtemperature.

The reaction is stopped by adding 100 μl of 1 M H₃PO₄ per well. Platesare read at 450 nm.

Results

The raw results and the cytokine release curves=f (LPS or complexconcentrations) do not allow comparison of different samples.Calculating the detoxification ratio can eliminate inter-blood donor andinter-test variability. Only the linear parts of the curves are takeninto account for calculation of the detoxification ratio. The maximumIL6 release beyond which a linear progression is no longer observed isdetermined and then, the amount of substance required to induced 50% ofthat maximum is calculated by linear regression.

The detoxification ratio is expressed as the ratio of the concentrationof the LPS-peptide complex inducing 50% of maximum IL6 release (ED₅₀expressed in pg/mL) in over that observed with LPS. Higher the ratio,stronger the detoxification is. As the detoxification ratio issystematically measured using whole blood of several independent donors,results are averaged.

The detoxification ratio observed with the LPS-peptide complex ismeasured several times. Mean data out of six values obtained in the IL6release assay: 64±20.

The IL6 release correlates with the TNFα and IL8 secretions. Therefore,the IL6 release assay is selected to routinely evaluate the inflammationdecrease observed with the LPS-peptide complex.

5.5. Conclusion

The detoxification ratio is measured between 10² and 10³, depending onthe test. The detoxification values are summarized in the followingtable.

LPS-peptide Detoxification Assays LPS L8 complex ratio LAL 25,000 EU/μg12-20 EU/μg 1,250-2,000 Limit pyrogen dose (IV) in rabbit 0.025 ng/kg10-25 ng/kg   400-1,000 Cytokine release test in mice IL6 = 25,000 pg/mLIL6 = 270 pg/mL 100 IL6 = 10,000 pg/mL IL6 = 100 pg/mL In vitro assay ofIL6 release by ED₅₀ = 2 pg/mL ED₅₀ = 880 pg/mL  64 human PBMC (ED₅₀:concentration of product inducing 50% of maximum IL6 release) LD50 ingalactosamine-sensitized 4 ng/souris 1 μg/souris (0.2-5) 250 mice

EXAMPLE 6 LPS Peptide Complex Stability Study

The stability of the LPS-peptide complex is studied for 6 months andevaluated by measuring the detoxification ratio in two assays (LAL andin vitro IL-6 release by huPBMC). Pyrogen test in rabbits may also beachieved.

6.1. In Vitro Stability of the Formulated LPS Peptide Complex

The stability of the formulated LPS-peptide complex is followed at 5°C., for 6 months. Measurements are made at day=0, 90 and 180 (6 months).Results are as follows.

Pyrogen test IL6 release from LAL assay Pyrogenic threshold, human bloodcells endotoxin as chosen: (detoxification ratio) (EU/μg) 10 ng/mL/kg IV3 6 3 6 3 6 0 months months 0 months months 0 months months 125 40 16314 58 10 C* C C C*: conform

The detoxification ratio in IL6 release test are not significantlydifferent after 3 and 6 months, indicating the stability of theLPS-peptide complex: LPS complexed with peptide remains detoxified after6 months at 5° C.

6.2. In Vitro Stability of the LPS Peptide Complex in PhysiologicalLiquid

The aim of the experiment is to verify that LPS is not released when thecomplex is administered and that the detoxification rate does notdecrease after a contact with a physiological liquid.

One mL of the LPS-peptide complex, mixed with 1 mL of human serum, isincubated at 37° C. The detoxification rate is evaluated after 1 and 24hours. Human serum and the LPS-peptide complex as prepared in section4.3. are also tested in parallel.

No significant difference of the detoxification evaluated by both assaysis observed after a 1-hour and 24-hour contact of the LPS-peptidecomplex with human serum at 37° C. and results are similar to theLPS-peptide complex control.

EXAMPLE 7 Immunogenicity of the LPS-Peptide Complex 7.1. BactericidalActivity of Anti LPS Antibodies Induced in Rabbits by the LPS-PeptideComplex

Immunization of three adult New-Zealand rabbits is performed with 100 μgof LPS-peptide complex by intramuscular (IM) and subcutaneous (SC)routes (2×0.5 mL and 5×0.2 mL respectively) in the presence of adjuvant.They receive three injections at 3-week interval; the first one withcomplete Freund adjuvant (FA), the second and third ones with incompleteFreund adjuvant. They are bled two weeks after the last injection. Acontrol group is immunized with the peptide with adjuvant (71 μg,equivalent to the amount of the peptide in 100 μg of LPS-peptidecomplex) using the same protocol.

The bactericidal activity of the serum (SBA) samples is evaluatedagainst the N. meningitidis strain used for LPS production as describedin Example 5 in the presence of baby rabbit serum as exogenous source ofcomplement.

SBA Assay

Sera are heat-inactivated during 30 min at 56° C. In the wells of a96-well microplate, heat-inactivated sera are then twofold seriallydiluted (10 times) in Dulbecco's phosphate buffered saline containingCa⁺⁺ and Mg⁺⁺ (volume per well: 50 μl).

Twenty five μl of a log phase culture of N. meningitidis grown inMueller-Hinton broth (4.10³ CFU/mL) and 25 μl of baby rabbit serum areadded to each well. The plate is incubated one hour at 37° C., undershaking.

Fifty μl of the mixture from each well are plated onto Mueller-Hintonagar. Petri dishes are incubated overnight at +37° C. in a 10% CO₂atmosphere.

In each experiment, controls include (i) bacteria and the complementsource without antibodies (complement control), (ii) bacteria andheat-inactivated complement, and (iii) bacteria and heat-inactivatedcomplement, in the presence of antibodies.

Bactericidal titre is reported as the highest reciprocal serum dilutionat which ≧50% killing of bacteria is observed as compared to thecomplement control.

SBA Results

Results are to be seen in the table hereinafter. High SBA titers areobtained with the complex. The specificity of the SBA response isconfirmed with the extinction of the response, when the sera (post-dose3) are adsorbed on LPS.

Post-dose 3 Post-dose 3 Rabbit Pre-immunized immunized immunized sera #sera sera adsorbed on LPS LPS-peptide A 16 512 4 complex B 16 1,024 8 C4 128 <4 Peptide D 4 16 4 E 16 16 87.2. Immune Response Induced in Mice with the LPS-Peptide Complex

Ten six-week old female outbred CD1 mice are immunized with a 10 μg doseof LPS-peptide complex by the subcutaneous route (0.2 mL). They receivetwo injections at 3-week interval. They are bled before each injectionand exsanguinated 14 days after the last injection. A control group isinjected with buffer.

In a first experiment, the antibody response is evaluated by ELISA andthe bactericidal activity of the post-dose 2 serum samples is evaluatedagainst the N. meningitidis stain used for LPS production as describedin Example 4 (homologous strain) and a heterologous N. meningitidisstrain [N. meningitidis group B strain RH873 (L4, 7, 8 immunotype)].

In a second experiment, the antibody response is evaluated by ELISA andthe opsonic activity of the post-dose 2 serum samples is evaluated byFACS.

7.2.1. Immunogenicity of LPS-Peptide Complex in Mice ELISA Titration ofAnti-LPS Antibodies

Wells of a 96-well microplate are coated with 100 μl of a 10 μg/mL LPSsolution in buffer 1 (PBS+10 mM MgCl₂). The plate is incubated 2 hoursat +37° C.; then overnight at +5° C.

The LPS solution is removed from the plate and wells are saturated with150 μl of buffer 2 (PBS+milk 1%+Tween 20 0.05%). The plate is incubatedone hour at 37° C.; then washed with buffer 3 (PBS+Tween 20 0.05%).

Sera are serially diluted 12-fold, directly in the wells using buffer 2(volume: 100 μl per well). The plate is incubated for 90 min at +37° C.;then washed with buffer 3.

Hundred μl of a diluted goat anti-mouse IgG (γ chain specific) or IgM (μchain specific) peroxydase conjugate are added in each well. The plateis incubated 90 min at 37° C. and then washed with buffer 3.

The reaction is developed by adding 100 μl of a tetramethylbenzidinesubstrate solution in each well. The plate is incubated 20 min at 37° C.The reaction is stopped by adding 1 M HCl and absorbance is measured at450 nm.

ELISA Results

Results are expressed in arbitrary ELISA Unit/mL (EU/mL) by comparisonto a reference serum.

In a preliminary immunization experiment, the ELISA assay is achievedusing a pool of 10 sera. As shown in the following table, theLPS-peptide complex is able to induce high anti-LPS IgG titers in miceand anti-LPS IgM after one injection (ELISA). A significant IgG boosteris observed after the second injection, whereas no significant IgMincrease is observed.

Anti-LPS IgG (EU/mL) Anti-LPS IgM (EU/mL) Post dose 1 Post dose 2 Postdose 1 Post dose 2 LPS-peptide 1,800 22,000 280 550 complex Buffer <40<40 <40 <40

In a further immunisation experiment, the ELISA assay is achievedindividually. After the second injection, seven out of the ten miceexhibits high IgG and IgM titers. Global mean titers expressed in logare about 3.7 and 2.8 respectively.

7.2.2. Bactericidal Activity of Mouse Sera

Bactericidal activity is measured as described in section 7.1.

Forty % of the post-dose 2 sera exhibit a bactericidal activity (SBAtitre≧16) against the homologous N. meningitidis strain. Four arebactericidal against the heterologous strain.

7.2.3. Opsonic Activity of Mouse Sera Opsonisation Assay

The opsonic activity is measured by flow cytometry technology (FACS)using human promyelocytic differentiated HL60 cells as effector and LPScoated latex fluorescent beads as target.

Effector cells are differentiated into granulocytes after treatment with100 mM dimethylformamide. The resulting cells are washed, resuspended inHanks' balanced salt solution and their concentration is adjusted to2.5×10⁷ cells/mL.

Sera are heat inactivated during 30 min at 56° C. In a 96 deep-wellmicroplate, heat-inactivated sera are serially fivefold diluted (3times) in Hanks balanced salt buffer containing Ca⁺⁺ and Mg⁺⁺ (volumeper well: 300 μl).

Twenty μl of LPS coated latex fluorescent beads and 10 μl of baby rabbitserum as exogenous complement source are added to each well. The plateis incubated 30 min at +37° C., under shaking.

Fifty μl of the effector cell suspension are added to each well. Theplate is incubated 30 min at +37° C., under shaking.

One hundred fifty μl from each well are transferred in a second deepwell and the reaction is stopped by adding 400 μl of PBS+0.02% EDTA. Theplate is centrifuged and washed twice with PBS+BSA buffer.

The phagocytosis of LPS coated beads by effector cells, in the presenceof antiserum and exogenous complement source is measured by FACS.

Opsonic activity is expressed as the inverse of serum dilution giving aphagocytosis product (PP)=200. PP is measured as the ratio number ofbeads/phagocytic cells×number of fluorescent cells.

Controls wells lacking antiserum and a positive monoclonal antiserum areincluded in each experiment.

Opsonisation Results

Eight out of ten mice exhibit high opsonic activity (≧350).

1-28. (canceled)
 29. A method of preparing an SAEP II peptide dimer offormula (I),NH₂-A-Cys1-B-Cys2-C-COOHNH₂-A′-Cys1-B′-Cys2-C′-COOH wherein Cys1 and Cys2 are each a cysteineamino acid; wherein the two Cys1 residues are linked together through anintermolecular disulphide bond and the two Cys2 residues are linkedtogether through an intermolecular disulphide bond; wherein A and A′independently are a peptide moiety of from 2 to 5 amino acid residues,in which at least 2 amino acid residues, are independently selected fromLys, Hyl (hydroxy-Lysine), Arg and His; wherein B and B′ independentlyare a peptide moiety of from 3 to 7 amino acid residues, which compriseat least two amino acid residues independently selected from Val, Leu,Ile, Phe, Tyr and Trp; and wherein C and C′ are optional and areindependently an amino acid residue or a peptide moiety of from 2 to 3amino acid residues; provided that the cationic amino acidresidues/hydrophobic amino acid residues ratio (cat/hydroph ratio) isfrom 0.4 to 2; the method comprising separating the dimer of formula (I)from (a) corresponding dimers of formula (II),NH₂-A-Cys1-B-Cys2-C-COOHHOOC-C′-Cys2-B′-Cys1-A′-NH₂ wherein the Cys1 residues are linked to theCys2 residues through intermolecular disulphide bonds; and/or (b)corresponding monomers of formulae NH₂-A-Cys1-B-Cys2-C-COOH andNH₂-A′-Cys1-B′-Cys2-C′-COOH, and/or (c) cyclic counterparts of thecorresponding monomers.
 30. The method according to claim 29, whereinthe SAEP II peptide dimer of formula (I) has a cat/hydroph ratio is from0.5 to 1.5.
 31. The method according to claim 30, wherein thecat/hydroph ratio is from 0.6 to
 1. 32. The method according to claim31, wherein the cat/hydroph ratio is from 0.6 to 0.8.
 33. The methodaccording to claim 29, wherein the B and B′ peptide moieties comprisethe sequence -X1-X2-X3-, in which X1 and X2; X2 and X3; or X1, X2 and X3are independently selected from Val, Leu, Ile, Phe, Tyr and Trp.
 34. Themethod according to claim 33 wherein the B and B′ peptide moietiescomprise: (i) the sequence -X1-X2-X3-, in which: X1 is Lys, Hyl, His orArg; X2 is Phe, Leu, Ile, Tyr, Trp or Val; and X3 is Phe, Leu, Ile, Tyr,Trp or Val; and (ii) amino acid residues, if any, each beingindependently selected from the group consisting of Val, Leu, Ile, Phe,Tyr, Trp, Lys, Hyl, Arg and His.
 35. The method according to claim 29,wherein the SAEP II peptide dimer of formula (I) is of formula (III)NH₂-A-Cys1-B-Cys2-COOHNH₂-A′-Cys1-B′-Cys2-COOH wherein the two Cys1 residues are linkedtogether through a disulphide bond and the two Cys2 residues are linkedtogether through a disulphide bond; and the SAEP II peptide dimer offormula (II) is of formula (IV)NH₂-A-Cys1-B-Cys2-COOHHOOC-Cys2-B′-Cys1-A′-NH₂, wherein the Cys1 residues are linked to theCys2 residues through a disulphide bond, and the monomers are offormulae NH₂-A-Cys1-B-Cys2-COOH or NH₂-A′-Cys1-B′-Cys2-COOH.
 36. Themethod of claim 29 wherein the SAEP II peptide dimer of formulae I is ahomologous peptide dimer.
 37. The method of claim 29 wherein the SAEP IIpeptide dimer of formula I is a parallel dimer of formula (VII)NH₂-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOHNH₂-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH wherein the two Cys1residues are linked together through a disulphide bond and the two Cys2residues are linked together through a disulphide bond, and the SAEP IIpeptide dimer of formula II is an antiparallel dimer form of formula(VI)NH₂-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOHCOOH-Cys2-Leu-Leu-Leu-Phe-Lys-Cys1-Lys-Thr-Lys-NH₂, wherein the Cys1residues are linked to the Cys2 residues through a disulphide bond, andand the monomers are of formulaeNH₂-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH andNH₂-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH.
 38. A method ofpreparing an SAEP II peptide dimer of formula (II),NH₂-A-Cys1-B-Cys2-C-COOHHOOC-C′-Cys2-B′-Cys1-A′-NH₂ wherein Cys1 and Cys2 are each a cysteineamino acid; wherein the Cys1 residues are linked to the Cys2 residuesthrough intermolecular disulphide bonds; wherein the two Cys1 residuesare linked together through an intermolecular disulphide bond and thetwo Cys2 residues are linked together through an intermoleculardisulphide bond; wherein A and A′ independently are a peptide moiety offrom 2 to 5 amino acid residues, in which at least 2 amino acidresidues, are independently selected from Lys, Hyl (hydroxy-Lysine), Argand His; wherein B and B′ independently are a peptide moiety of from 3to 7 amino acid residues, which comprise at least two amino acidresidues independently selected from Val, Leu, Ile, Phe, Tyr and Trp;and wherein C and C′ are optional and are independently an amino acidresidue or a peptide moiety of from 2 to 3 amino acid residues; providedthat the cationic amino acid residues/hydrophobic amino acid residuesratio (cat/hydroph ratio) is from 0.4 to 2; the method comprisingseparating the dimer of formula (I) from (a) corresponding dimers offormula (I),NH₂-A-Cys1-B-Cys2-C-COOHNH₂-A′-Cys1-B′-Cys2-C′-COOH wherein Cys1 and Cys2 are each a cysteineamino acid; and/or (b) corresponding monomers of formulaeNH₂-A-Cys1-B-Cys2-C-COOH and NH₂-A′-Cys1-B′-Cys2-C′-COOH, and/or (c)cyclic counterparts of the corresponding monomers.
 39. The methodaccording to claim 38, wherein the SAEP II peptide dimer of formula (II)has a cat/hydroph ratio is from 0.5 to 1.5.
 40. The method according toclaim 39, wherein the cat/hydroph ratio is from 0.6 to
 1. 41. The methodaccording to claim 40, wherein the cat/hydroph ratio is from 0.6 to 0.8.42. The method according to claim 38, wherein the B and B′ peptidemoieties comprise the sequence -X1-X2-X3-, in which X1 and X2; X2 andX3; or X1, X2 and X3 are independently selected from Val, Leu, Ile, Phe,Tyr and Trp.
 43. The method according to claim 42 wherein the B and B′peptide moieties comprise: (i) the sequence -X1-X2-X3-, in which: X1 isLys, Hyl, His or Arg; X2 is Phe, Leu, Ile, Tyr, Trp or Val; and X3 isPhe, Leu, Ile, Tyr, Trp or Val; and (ii) amino acid residues, if any,each being independently selected from the group consisting of Val, Leu,Ile, Phe, Tyr, Trp, Lys, Hyl, Arg and His.
 44. The method according toclaim 38, wherein the SAEP II peptide dimer of formula (I) is of formula(III)NH₂-A-Cys1-B-Cys2-COOHNH₂-A′-Cys1-B′-Cys2-COOH wherein the two Cys1 residues are linkedtogether through a disulphide bond and the two Cys2 residues are linkedtogether through a disulphide bond; and the SAEP II peptide dimer offormula (II) is of formula (IV)NH₂-A-Cys1-B-Cys2-COOHHOOC-Cys2-B′-Cys1-A′-NH₂, wherein the Cys1 residues are linked to theCys2 residues through a disulphide bond, and the monomers are offormulae NH₂-A-Cys1-B-Cys2-COOH or NH₂-A′-Cys1-B′-Cys2-COOH.
 45. Themethod of claim 38 wherein the SAEP II peptide dimer of formulae II is ahomologous peptide dimer.
 46. The method of claim 38 wherein the SAEP IIpeptide dimer of formula II is an antiparallel dimer form of formula(VI)NH₂-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOHCOOH-Cys2-Leu-Leu-Leu-Phe-Lys-Cys1-Lys-Thr-Lys-NH₂, wherein the Cys1residues are linked to the Cys2 residues through a disulphide bond, andthe SAEP II peptide dimer of formula I is a parallel dimer of formula(VII)NH₂-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOHNH₂-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH wherein the two Cys1residues are linked together through a disulphide bond and the two Cys2residues are linked together through a disulphide bond, and the monomersare of formulae NH₂-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH andNH₂-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH.